This project is in response to program announcement NIH PA-08-110 titled New Technologies for Transient Molecular Complex Characterization. Transient protein-protein interactions play an important role in regulating physiological functions in cells, including gene expression, transport, signal transduction, and cell cycle control. However, a very limited repertoire of analytical methods is available for studying such interactions. A robust methodology for studying transient protein-protein interactions would provide critical information for the development of diagnostic methods/agents and discovery of novel treatments. The primary goal of this application is to strategically design and develop carbene-generating photocrosslinking reagents, with quantitative mass spectrometric detection and identification elements, for the systematic analysis of transient protein-protein interactions. The final objective of this project is to develop a kit(reagent)-method-software package for the identification of amino acid residues involved in transient protein-protein interactions. Two sets of photocrosslinking reagents (oxLink I and II) with a reversible linkage and an MS signature ion segment have been designed. Proteins of interest will be labeled with photocrosslinking reagents. Upon photolysis, carbene generated from oxLink will form a covalent bond with the interacting proteins. The crosslinked proteins will be digested and the reversible linkage will allow efficient purification/enrichment of the crosslinked peptides using solid phase extraction. Upon fragmentation, these peptides will generate the b, y, and signature ions for the identification and quantitation of crosslinked products. In phase I, (a) the reversible link formation and cleavage conditions will be optimized using model peptides as substrates, (b) purification/enrichment methods will be developed using bovine serum albumin (BSA) with reversible linkage as a model system, (c) an oxLink set will be synthesized, and (d) the MS fragmentation pattern of the model crosslinked products using oxLink will be tested. In phase II, the combination of the above approaches will be tested and refined iteratively using rhodopsin and transducin as a model system. General methodology for detecting transient protein-protein interactions and structural studies of transient protein complexes will be developed in collaboration with several academic research groups serving as alpha testing sites. A database compiling the oxLink modified amino acid residues will be initiated, as well as the development of related software tools for MS analysis and molecular modeling. PUBLIC HEALTH RELEVANCE: Transient protein-protein interactions are essential for the biological actions of nearly all cellular functions, including membrane proteins, which are the target of half of all drug development efforts in the existing pharmaceutical market. Despite their importance, little information is available about membrane protein-protein interactions at the atomic level. This proposal aims to continue the development of a novel approach for determining transient protein complex structures using photocrosslinking, mass spectroscopy, and molecular modeling. This approach can serve as a powerful platform for the discovery of novel treatment strategies and rational design of new drug candidates.